Heterocyclic dioxethane substrates, process for their preparation and their use

ABSTRACT

Compounds of the general formula Ia and Ib are described ##STR1## in which R 1  and R 2  are the same or different and represent hydrogen, straight-chain or branched C 1  to C 6  lower alkyl or an aryl group optionally substituted by electron-withdrawing groups, R 3  denotes a cleavable group, W is hydrogen, halogen or a pseudohalogen and at least one of the groups R 4  or R 5  is a group stabilizing the dioxetane structure and at most one of the groups R 4  or R 5  represents hydrogen and X or Y represents oxygen, N-R or C(R) 2  in which R has the meanings stated for R 1  and R 2  or represents a mesomeric double bond or a carbonyl group and n denotes the number 0 or 1 and m denotes the number 1 or 2, as well as a process for their production. These compounds are new and can be used as substrates in immunological assays and in DNA diagnostics using activating agents for colour formation.

This application is a 371 of PCT/EP96/04506 Oct. 17, 1996.

The invention concerns heterocyclic dioxetane substrates, processes fortheir production and their use in enzymatic analytical methods.

It has been known for a long time that oxiluciferin is formed by thereaction of luciferin with luciferase, oxygen and ATP. In this reactionlight (wavelength maximum at 562 nm) is emitted as chemiluminescence. Inthis process a dioxetane is presumably formed as an energy-richintermediate product (F. McCapra, Chem. Commun. 155 (1968)). Numerouschemiluminescent 1,2-dioxetane compounds have been developed based onthis postulate.

The adamantyl residue has been described for the stabilization of theunstable 1,2-dioxetanes (EP-A 0 254 051, EP-A 0 352 713, WO 91/03479, WO90/07511, WO 92/04341 as well as publications cited therein). Thechemiluminescence yield then develops with the decay of the dioxetanesand is dependent among others on the fluorescent properties of theemitter formed. Although the phenolates that are formed on decay of thedioxetane derivatives that are usually used(3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetaneor corresponding dioxetanes halogenated on the adamantyl residue) have agood fluorescence quantum yield, the substrates have an overall lowchemiluminescence. Dioxetanes with improved chemiluminescence yieldswould be particularly desirable in the triggered decay of dioxetanes.

Stabilized dioxetane derivatives are known from WO 93/20083 which decayinto oxyluciferin and phenylthiazoline derivatives as emitters. Althoughthese dioxetanes decay with a higher light yield than other knowndioxetanes, they are very difficult to prepare synthetically.

Hence the object of the present invention was to provide new1,2-dioxetanes which are stable and which only decay by reaction with anactivating agent with the formation of a high light yield and which canbe prepared by relatively simple chemical synthesis. This object isachieved by a compound of the general formula Ia or Ib ##STR2## in whichR¹ and R² are each the same or different and represent hydrogen,straight-chain or branched C₁ to C₆ lower alkyl, individually ortogether a 3-6 carbon atom cycloalkyl or an aryl group (preferablyphenyl or naphthly with optionally one or several substitutedelectron-withdrawing groups provided that only one of the residues R¹ orR² is hydrogen,

m denotes the number 1 or 2,

R³ is a cleavable group which can be cleaved off by an activating agent,

W is hydrogen, halogen or a pseudohalogen such as rhodanide or cyanideresidues and is preferably in the ortho position relative to the O-R³group,

at least one of the groups R⁴ or R⁵ is a group stabilizing the dioxetanestructure and at most one of the groups R⁴ or R⁵ represents hydrogen,

and X and Y represent oxygen, N-R or C(R)₂ in which R has the statedmeanings for R¹ and R² or it is a double bond in a mesomeric system orit represents a carbonyl group and n denotes the number 0 or 1.

Acids, bases, salts, enzymes, inorganic or organic catalysts andelectron donors are preferably used as activating agents.

The group O-R³ can be located at any position of the phenyl ring. If Xis oxygen, the group O-R³ is preferably located in the 5-position in thecase of compounds of the general formula Ia and where n is 0, in thecase of Ib and where m equals 1 it is preferably in the 3-position andin the case of Ib and where m equals 2 it is preferably in the4-position.

The group O-R³ is preferably a hydroxy salt or an oxy acid, phosphate,arylcarboxyl ester or alkylcarboxyl ester, alkyloxy, alkylsilyloxy orarylsiloxy, sulfate, oxypyranoside or a glyceridyl or phosphoryl residueor a steroid derivative. Phosphate and a dimethyl-tertiarybutyl-silyloxy group are particularly preferred for O-R³.

The composition of the aryl and alkyl residues is not critical.Preferred aryl in this application are phenyl and naphthyl, andpreferred alkyl residues are C₁ -C₆, unless otherwise noted. Any personskilled in the art can select suitable residues for R³ withoutdifficulty. The only requirements are solubility and cleavability of R³by the activating agents. Any reagent which is capable of removing theR³ group, and therefore provide an activated phenoxide ion whichdecomposes with light emission can be used as the activating agent. Theparticular type of the reagent depends upon the nature of the R³ group;e.g. if R³ is hydrogen, the reagent is a base; if SiR₃, the reagent is afluoride anion; if R³ is PO⁻ ₃, the reagent is a phosphatase enzyme, andso on.

Hydrogen, methyl, ethyl or phenyl groups are preferred for R¹ and R², orif R¹ and R² together denote a phenylene group with (C)_(m) and m equals2 whereby the phenyl and phenylene groups can for example be substitutedby a halogen residue. If for example phosphate is used as O-R³, thechemiluminescence reaction can be induced by adding alkalinephosphatase. If a galactoside is used the chemiluminescence reaction canbe induced by β-galactosidase. If a silyloxy residue is used as O-R³,the chemiluminescence can be induced by adding fluoride.

Suitable groups R⁴ and/or R⁵ that stabilize the dioxetane structure aregroups which protect the dioxetane group from uncontrolled conversion.This stabilization is preferably achieved by steric shielding of thisgroup. Hence condensed aliphatic or aromatic ring systems which canoptionally be substituted by electron withdrawing groups come intoparticular consideration for this purpose. Preferred systems are 2- to 5-membered cycloalkyl with 3-6 carbon atoms; preferably at least one ofthe members is an aromatic ring. Carbonyl or halogen can for example beused as electron withdrawing groups. However, all groups with a positiveσ_(p) value can be used (See March, Advanced Organic Chemistry, WileyInterscience 1992, 4th Ed. p. 280). Preferred groups in this regard arehalogen, cyano and RCO groups where R is C₁ -C₆ alkyl. Adamantanyl,phenyl, cyclohexyl, secondary and tertiary aliphatic alkyl groups (suchas e.g. the t-butyl group) in a substituted and unsubstituted form arealso suitable. In this case R⁴ and R⁵ can be the same or different. Inthe case of phenyl and/or cyclohexyl groups, individual residues as wellas in the form of condensed ring systems come into consideration assubstituents which can optionally have further structural units such ase.g. a cyclopentyl residue as in steroids. Corresponding residues aresummarized within the scope of the present invention as polycycloalkyland polycycloaryl residues. In the case of the adamantyl residue or forexample a steroidyl residue, this is preferably bound in such a way thatR⁴ and R⁵ denote parts of the ring structure and are consequentlybridged (formulae Ia' and Ia"): ##STR3## Appropriate steriod derivativesfor R⁴ and R⁵ are as follows: ##STR4## in which each of A, B, C is H; Aor B is halogen; A or C is methyl; each of X, Y can be H, ═O, ═CHO₂ Et,CH(Me)CH₂ OAc,₋₋ CH(Me)CO₂ Me, OAc, C═CH CH(Me)CH₂ OH, CH(Me)CO₂ H, OH,COCH₂ OAc, OCH(Me)CH₂ OEt or both together are OCH₂ OCH₂ O, N--OH,NNHPh, NNMe₂, NNHCSNH₂ or NNHCONH₂ ; D is OH or a protected OH or ═O; Eis halogen, e.g. fluor, chlor; and Z can be alkyl consisting of 1 to 10C atoms. See also, D. Weiss et al., Synthesis, August 1992, p. 751-752,hereby incorporated by reference.

According to the invention the dioxetanes of benzofuranes have proven tobe particularly suitable:

3-(7'-t.-butyldimethylsilyloxy-2',2'-dimethylbenzofuran-3'-yl)-4-spiro-(tricyclo[3.3.1.1.³,7]yl)-1,2-dioxetane, the disodium salt of3-(7'-phosphoryl-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(tricyclo[3.3.1.1.³,7 ]yl)-1,2-dioxetane,3-(7'-t.-butyl-dimethylsilyloxy-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7]yl)-1,2-dioxetane, the disodium salt of3-(7'-phosphoryl-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7 ]-yl)-1,2-dioxetane,3-(5'-t.-butyldimethylsilyloxy-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7]yl)-1,2-dioxetane, the disodium salt of3-(5'-phosphoryl-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7 ]yl)-1,2-dioxetane,3-(5'-t.-butyldimethylsilyloxy-6'-chloro-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7]yl)-1,2-dioxetane, the disodium salt of3-(5'-phosphoryl-6'-chloro-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7 ]yl)-1,2-dioxetane,(4'Ξ)-5"-t.-butyldimethylsilyloxy-6"-chloro-2",2"-dimethyl-17-oxo-(3.zeta.O)-dispiro[androsta-1,4-diene-3,3'-[1,2]dioxetane-4',3"-benzofuraneor(4Ξ)-6"-chloro-2",2"-dimethyl-17-oxo-(3ξ)-dispiro[androsta-1,4-diene-3,3'-[1,2]dioxetane-4',3"-benzofuran-5"-yldisodium phosphate.

The compounds of the general formulae Ia and Ib are new. A process forsynthesizing these compounds has not been known previously.

Hence a subject matter of the invention is also a process for theproduction of the compounds of the general formulae Ia and Ib, which ischaracterized in that heterocyclic carbonyl compounds of the generalformulae II, III or IV ##STR5## in which R⁸ represents a straight-chainor branched alkyl residue with 1 to 6 C atoms, preferably a methylresidue or a hydrogen atom, W represents a halogen or pseudohalogenresidue such as for example SCN or CN or represents a hydrogen atom, Xrepresents oxygen or N-alkyl and the residues R¹ and R² have themeanings mentioned above are reacted with a compound of the generalformula (V), ##STR6## in which R⁴ and R⁵ have the above-mentionedmeanings, with the exclusion of water and air in the presence oftitanium trichloride and a reducing agent, if R⁸ is alkyl, the reactionproduct is dealkylated in the presence of for example boron triiodide orsodium ethanethiolate, or if R⁸ denotes hydrogen, the cleavable group R³is introduced directly according to a method familiar to a personskilled in the art and subsequently dioxygenated to form dioxetane.

The heterocyclic carbonyl compounds are produced according to methodsdescribed in the literature cf. e.g. Tetrahedron 1978, 34, 2035; Helv.Chim. Acta 1972, 55, 1567; J.Agric. Food. Chem. 1968, 300; J. Am. Chem.Soc. 1967, 87, 6527.

The reaction of the compounds II, III and IV with the compound V iscarried out in a solvent that is as unpolar as possible, preferablytetrahydrofuran or dimethoxyethane, in the presence of titaniumtrichloride and a reducing agent, preferably zinc/copper or lithiumaluminium hydride. Aldehydes and ketones that are especially suitable ascompound V are those which have residues R⁴ and R⁵ that stabilize thefinal product the dioxetane. Such a stabilization can be achieved bysteric shielding of the dioxetane structure. Hence suitable ketones andaldehydes are for example adamantanone, polycyclic ketones such as e.g.a steroid ketone, secondary and tertiary aliphatic ketones and aldehydessuch as e.g. t.-butylketone. These ketones can carry furthersubstituents preferably chlorine atoms.

In the case of compounds in which R⁸ is an alkyl residue preferablymethyl, it is expedient to carry out the dealkylation with adealkylation agent preferably with boron triiodide or sodiumethanethiolate.

It is also possible to convert the heterocyclic ketones II, III and IVinto the heterocyclic thiones of the general formulae VI, VII and VIIIin which R⁸ and W, X and R¹ and R² represent the above-mentionedsubstituents, and subsequently to react them with diazo compounds of thegeneral formula IX in which R⁴ and R⁵ have the above-mentioned meanings.In the case that R⁸ is methyl the alkenes that are formed aredealkylated according to the method described above. Subsequently thecleavable group R³ is introduced as described above and thedioxygenation is carried out. ##STR7##

The carbonyl function is converted into a thiocarbonyl function byreacting the heterocyclic ketones with a sulfurization reagent,preferably Lawesson reagent(2,4-bis-(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide)or phosphorus pentasulfide in toluene or pyridine.

The reaction with diazo components for example with di-tertiarybutyldiazomethane or with a diazosteroid is preferably carried out in anunpolar solvent such as diethyl ether. The diazo component is producedby oxidation of hydrazones with an oxidizing agent preferably manganesedioxide or silver oxide. Diazo compounds are preferably suitable ascompound IX which stabilize the dioxetane by means of the residues R⁴and R⁵. Such a stabilization can be achieved by steric shielding of thedioxetane structure. Hence suitable diazo compounds are for examplediazoadamantane, chlorodiazoadamantane, diphenyldiazomethane, polycyclicdiazo compounds such as e.g. a diazosteroid, secondary or tertiaryaliphatic ketones and aldehydes such as e.g. di-tert.-butyl ketone.These diazo compounds can carry further substituents preferably chlorineatoms.

The cleavable chemically labile group is introduced by methods familiarto a person skilled in the art. Such methods are for example describedin Houben Weyl XII/2.

It is expedient to carry out the dioxygenation by reacting the alkenewhich is either dissolved in methylene chloride or in methanol at atemperature of about -30 to -10° C. with singlet oxygen which is formedin situ by irradiation with visible light using a sensitizer (RoseBengal, methylene blue, cf.Tetrahedron Letters 1988, 3137-3140).

The purification is carried out in particular by filtration or othermethods familiar to a person skilled in the art.

A further subject matter of the invention is a method for thedetermination of an acid, base, a salt, enzyme, inorganic or organiccatalyst and electron donors by reacting this compound with a compoundof the general formula Ia or Ib and measuring the emitted light as ameasure of the content of the compound to be determined.

This method is particularly preferably used to determine enzymes,especially marker enzymes, in immunological systems or for DNAdiagnostics with labelled DNA probes. A detailed description of themethod involved can be found in Kessler, Springer, Publ. Berlin 1992, inparticular chapter 3.2.12, .13 and 19.6.

Alkaline phosphatase is preferably determined in which case thechemically labile group R³ is phosphate or β-galactosidase in which caseR³ is a galactoside.

The invention is elucidated by the following examples:

EXAMPLE 1 2-(4'-Methoxyphenyl)-4H-1,3-benzoxazin-4-one

2.5 ml pyridine is added to a suspension of 0.25 mol salicylamide in 50ml xylene. While boiling under reflux (water separator) 0.26 mol4-methoxybenzoyl chloride is added within 3 hours and it is heated toboiling for a further 3 hours under reflux. A further 2.5 ml pyridine isadded and it is heated for a further 8 hours. After cooling to roomtemperature it is poured onto 400 ml isopropanol and suction filtered.The residue is recrystallized from ethylene glycol monomethyl ether.

Yield 0.2 mol

EXAMPLE 2 4-Adamantylidene-2-(4'-methoxyphenyl)-4H-1,3-benzoxazine

21 mmol lithium aluminium hydride is carefully added to a suspension of40 mmol titanium chloride in 100 ml absolute THF while stirringvigorously. After stirring for 1 hour at room temperature, a mixture of15 mmol 2-(4'-methoxyphenyl)-4H-1,3-benzoxazine-4-one and 11 mmoladamantanone is added while cooling on ice. It is heated to boiling for60 min under reflux. After cooling to room temperature it is poured onto200 ml water. The mixture is shaken out twice with 200 ml ethyl acetateeach time. The combined organic phases are washed once with water andseparated. After drying over sodium sulfate it is filtered and thesolvent is removed by distillation. The oily residue is separated bycolumn chromatography (silica gel: ethyl acetate/petroleum ether 1:9).

Yield 6 mmol

EXAMPLE 3 4-Adamantylidene-2-(4'-hydroxyphenyl)-4-H-1,3-benzoxazine

A solution of 6 mmol4-adamantylidene-2-(4'-methoxyphenyl)-4H-1,3-benzoxazine in 20 mlabsolute methylene chloride is added dropwise to a solution of 12 mmolboron triiodide while stirring at -25° C. with the exclusion of air andwater. The mixture is heated to room temperature within one hour andstirred for a further 4 hours. After admixing with 50 ml water theorganic phase is separated and dried over sodium sulfate. The solvent isremoved by distillation. The oily residue is separated by columnchromatography (silica gel: ethyl acetate/petroleum ether 1:3).

Yield 3.2 mmol

EXAMPLE 44-Adamantylidene-2-(4'-t.butyldimethylsilyloxyphenyl)-4H-1,3-benzoxazine

3 mmol 4-adamantylidene-2-(4'-hydroxyphenyl)-4H-1,3-benzoxazine issuspended in 30 ml absolute methylene chloride. 6 mmol imidazole and 6mmol t.-butyldimethylsilyl chloride are added in succession. Afterstirring for 14 h at room temperature it is filtered. The filtrate isshaken out successively with 20 ml 1 M sodium hydroxide solution, 20 ml2 M hydrochloric acid and 20 ml water each time. The separated organicphase is dried over sodium sulfate. The solvent is removed bydistillation. The oily residue is recrystallized from a small amount ofpetroleum ether.

Yield 2.7 mmol

EXAMPLE 5 Disodium salt of4-adamantylidene-2-(4'-phosphoryl-phenyl)-4H-1,3-benzoxazine

A solution of 3 mmol4-adamantylidene-2-(4'-hydroxyphenyl)-4H-1,3-benzoxazine in a mixture of3 ml acetone and 3 ml pyridine is added dropwise to a solution of 1.5 mlphosphoroxy chloride in 7.5 ml acetone while stirring at 0° C. It isstirred for 1 hour at 0° C. and subsequently poured onto 60 ml saturatedsaline solution and suction filtered. The residue is suspended in 1.5 mlwater. A pH value of 10 is set with 2 M sodium hydroxide solution. It isadmixed with 30 ml ethanol and suction filtered. The residue is dried ina vacuum.

Yield 2.2 mmol

EXAMPLE 63-[2'-(4"-t.butyldimethylsilyloxyphenyl)-4'H-1,3-benzoxazin-4'-yl]-4-spiroadamantyl-dioxetane

2 mmol4-adamantylidene-2-(4'-t.butyldimethylsilyloxyphenyl)-4H-1,3-benzoxazinedissolved in 20 ml absolute methylene chloride is irradiated for 2 hoursat -30° C. with a 1000 watt sodium-vapour lamp in the presence of 0.01mol % immobilized Rose Bengal while passing in dry oxygen. The reactionproceeds quantitatively. The sensitizer can be removed by filtration.The filtrate contains the dioxetane.

Yield 2 mmol

EXAMPLE 7 Disodium salt of3-[2'-(4"-phosphoryl-phenyl)-4'H-1,3-benzoxazin-4'-yl]-4-spiroadamantyl-dioxetane

2 mmol of the disodium salt of4-adamantylidene-2-(4'-phosphoryl-phenyl)-4H-1,3-benzoxazine dissolvedin 20 ml absolute methanol is irradiated for 2 hours at -30° C. with a1000 watt sodium-vapour lamp in the presence of 0.01 mol % immobilizedRose Bengal while passing in dry oxygen. The reaction proceedsquantitatively. The sensitizer can be removed by filtration. Thefiltrate contains the dioxetane.

Yield 2 mmol

EXAMPLE 8 2-(3'-Methoxyphenyl)-5,5-dimethyl-oxazolin-4-one

50 mmol acetone is added at 0° C. to a solution of 57 mmol potassiumcyanide in 50 ml water. 57 mmol 3-methoxybenzoyl chloride is addeddropwise within 90 minutes at room temperature while stirringvigorously. After stirring for a further 90 min at 0° C. it is allowedto stand for 10 h at room temperature. It is admixed with 100 ml diethylether and the mixture was shaken out twice with 50 ml saturated sodiumhydrogen carbonate solution each time and then with 100 ml water. Theorganic phase is separated and dried over sodium sulfate. The solvent isremoved by distillation. The oily residue (6.6 g 3-methoxybenzoicacid-2'-cyanoisopropyl ester) is dried in a high vacuum and subsequentlyintroduced into a mixture of 52 ml acetanhydride containing 10.4 ml 35%aqueous tetrafluoroboric acid. It is heated for 1 hour to boiling underreflux and after cooling to room temperature it is admixed with 300 mlether. The precipitate that forms is suspended in 70 ml toluene. 70 mmoltriethylamine is added to the suspension and it is stirred for 1 hour atroom temperature. It is filtered. The filtrate is concentrated todryness in a vacuum. The oily residue is separated by columnchromatography (silica gel: ethyl acetate/petroleum ether 1:1).

Yield 17 mmol

EXAMPLE 9 4-Adamantylidene-2-(3'-methoxyphenyl)-5,5-dimethyl-oxazoline

122 mmol zinc-copper couple is added to a suspension of 35 mmol titaniumtrichloride in 60 ml absolute THF while stirring carefully. Afterheating for one hour under reflux a mixture of 11.4 mmol2-(3'-methoxyphenyl)-5,5-dimethyl-oxazolin-4-one and 8.1 mmoladamantanone is added after cooling to room temperature. It is heatedfor 4 h to boiling at reflux. After cooling to room temperature it ispoured onto 200 ml water. The mixture is shaken out twice with 200 mlethyl acetate. The combined organic phases are washed once with waterand separated. After drying over sodium sulfate it is filtered and thesolvent is removed by distillation. The oily residue is separated bycolumn chromatography (silica gel: ethyl acetate/petroleum ether 1:9).

Yield 7 mmol

EXAMPLE 10 4-Adamantylidene-2-(3'-hydroxyphenyl)-5,5-dimethyl-oxazoline

A solution of 7 mmol4-adamantylidene-2-(3'-methoxyphenyl)-5,5-dimethyl-oxazoline in 20 mlabsolute methylene chloride is added dropwise to a solution of 14 mmolboron triiodide while stirring at -25° C. with the exclusion of air andwater. The mixture is heated to room temperature within one hour andstirred for a further 4 h. After admixing with 50 ml water the organicphase is separated and dried over sodium sulfate. The solvent is removedby distillation. The oily residue is separated by column chromatography(silica gel: ethyl acetate/petroleum ether 1:3).

Yield 3.9 mmol

EXAMPLE 114-Adamantylidene-2-(3'-t.butyldimethylsilyloxyphenyl)-5,5-dimethyl-oxazoline

3 mmol 4-adamantylidene-2-(3'-hydroxyphenyl)-5,5-dimethyl-oxazoline issuspended in 30 ml absolute methylene chloride. 6 mmol imidazole and 6mmol t.-butyldimethylsilyl chloride are added successively. Afterstirring for 14 h at room temperature it is filtered. The filtrate issuccessively shaken out with 20 ml 1 M sodium hydroxide solution, 20 ml2 M hydrochloric acid and 20 ml water each time. The separated organicphase is dried over sodium sulfate. The solvent is removed bydistillation. The oily residue is separated by column chromatography(silica gel: petroleum ether).

Yield 2.5 mmol

EXAMPLE 12 Disodium salt of4-adamantylidene-2-(3'phosphorylphenyl)-5,5-dimethyl-oxazoline

A solution of 3 mmol4-adamantylidene-2-(3'-hydroxyphenyl)-5,5-dimethyl-oxazoline in amixture of 3 ml acetone and 3 ml pyridine is added dropwise to asolution of 1.5 ml phosphoroxy chloride in 7.5 ml acetone while stirringat 0° C. It is stirred for 1 h at 0° C. and subsequently poured onto 60ml saturated saline solution and suction filtered. The residue issuspended in 1.5 ml water. A pH value of 10 is set using 2 M sodiumhydroxide solution. It is admixed with 30 ml ethanol and aspirated. Theresidue is dried in a vacuum.

Yield 2.0 mmol

EXAMPLE 133-[2'-(3"-t.butyldimethylsilyloxyphenyl)-5',5'-dimethyl-oxazoline-4'-yl]-4-spiroadamantyl-dioxetane

2 mmol4-adamantylidene-2-(3I-t.-butyldimethylsilyloxyphenyl)-5,5-dimethyl-oxazolinedissolved in 20 ml absolute methylene chloride is irradiated for 2 h at-30° C. with a 1000 watt sodium-vapour lamp in the presence of 0.01%immobilized Rose Bengal while passing in dry oxygen. The reactionproceeds quantitatively. The sensitizer can be removed by filtration.The filtrate contains the dioxetane.

Yield 2 mmol

EXAMPLE 14 Disodium salt of 3-[2'-(3"phosphorylphenyl)-5',5'-dimethyl-oxazoline-4'-yl]-4-spiroadamantyl-dioxetane

2 mmol disodium salt of4-adamantylidene-2-(3'phosphorylphenyl)-5,5-dimethyl-oxazoline dissolvedin 20 ml absolute methanol is irradiated for 2 hours at -30° C. with a1000 watt sodium-vapour lamp in the presence of 0.01% immobilized RoseBengal while passing in dry oxygen. The reaction proceedsquantitatively. The sensitizer can be removed by filtration. Thefiltrate contains the dioxetane.

Yield 2 mmol

EXAMPLE 15 7-Hydroxy-2,2-dimethyl-benzofuran-3-one

7-Hydroxy-2,3-dihydro-2,2-dimethylbenzofurane is dissolved in 100 mlpyridine. 0.5 mol benzoyl chloride is added dropwise at 0° C. within 4h. Subsequently it is stirred for 10 h at room temperature. It is pouredonto 200 ml ice water and aspirated. The residue is washed with 500 ml 2M hydrochloric acid and then with 1000 ml water and dried in air. Asolution of 0.906 mol chromium trioxide in 370 ml glacial acetic acid isadded dropwise to a solution of this crude product (0.45 mol) in 600 mlglacial acetic acid in such a way that the temperature of the mixturedoes not exceed 20° C. After the dropwise addition is completed it isstirred for a further 1 hour at 40° C. and subsequently poured onto 2 lwater. It is suction filtered. The residue is washed with 1 l water andrecrystallized from ethanol/water 3:1. The benzoyl compound is fed into400 ml 10% ethanolic potassium hydroxide solution and the mixture isheated for 1 hour to 70° C. It is admixed with 3 l water and suctionfiltered. The residue is recrystallized from 2 l water.

Yield 0.16 mol

EXAMPLE 163-(5'-Chloro-tricyclo[3.3.1.1.3.7]yl)-7-hydroxy-2,2-dimethyl-benzofurane

550 mmol zinc-copper-couple is carefully added to a suspension of 158mmol titanium trichloride in 120 ml absolute THF while stirringvigorously. After heating for 1 hour under reflux, a mixture of 52 mmol7-hydroxy-2,2-dimethyl-benzofuran-3-one and 37 mmol5-chloro-adamantan-2-one is added at room temperature. It is heated for30 min under reflux to boiling. After cooling to room temperature it ispoured onto 200 ml water. The mixture is shaken out twice with 200 mlethyl acetate each time. The combined organic phases are washed oncewith water and separated. After drying over sodium sulfate it isfiltered and the solvent is removed by distillation. The oily residue isseparated by column chromatography (silica gel: ethyl acetate/petroleumether 1:9).

Yield 30 mmol

EXAMPLE 17 3-(5'-chloro-tricyclo[3.3.1.1.³,7]-7-t.-butyldimethylsilyloxy-2,2-dimethyl-benzofurane

30 mmol 3-(5'-chloro-tricyclo[3.3.1.1.³,7]yl)-7-hydroxy-2,2-dimethyl-benzofurane is suspended in 30 ml absolutemethylene chloride. 60 mmol imidazole and 60 mmol t.-butyldimethylsilylchloride are added in succession. After stirring for 14 h at roomtemperature it is filtered. The filtrate is shaken out successively with100 ml 1 M sodium hydroxide solution, 100 ml 1 M hydrochloric acid and20 ml water in each case. The separated organic phase is dried oversodium sulfate. The solvent is removed by distillation. The oily residueis separated by column chromatography (silica gel: petroleum ether).

Yield 27 mmol

EXAMPLE 18 Disodium salt of 3-(5'-chloro-tricyclo[3.3.1.1.³,7]yl)-7-phosphoryl-2,2-dimethyl-benzofurane

A solution of 30 mmol 3-(5'-chloro-tricyclo[3.3.1.1.³,7]yl)-7-hydroxy-2,2-dimethyl-benzofurane in a mixture of 30 ml acetoneand 30 ml pyridine is added dropwise at 0° C. to a solution of 15 mlphosphoroxy chloride in 75 ml acetone while stirring. It is stirred for1 hour at 0° C. and subsequently poured onto 600 ml saturated salinesolution and suction filtered. The residue is suspended in 15 ml water.A pH of 10 is set using 2 M sodium hydroxide solution. It is admixedwith 300 ml ethanol and suction filtered. The residue is dried in avacuum.

Yield 23 mmol

EXAMPLE 193-(7'-t.-butyldimethylsilyloxy-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7]yl)-1,2-dioxetane

10 mmol 3-(5'-chloro-tricyclo[3.3.1.1.³,7]yl)-7-t.-butyldimethylsilyloxy-2,2-dimethyl-benzofurane dissolved in200 ml absolute methylene chloride is irradiated for 2 hours at -30° C.with a 1000 watt sodium-vapour lamp in the presence of 0.01 mol %immobilized Rose Bengal while passing in dry oxygen. The reactionproceeds quantitatively. The sensitizer can be removed by filtration.The filtrate contains the dioxetane.

Yield 20 mmol

EXAMPLE 20 Disodium salt of3-(7'-phosphoryl-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7]yl)-1,2-dioxetane

2 mmol disodium salt of 3-(5'-chloro-tricyclo [3.3.1.1.³,7]yl)-7-phosphoryl-2,2-dimethyl-benzofurane dissolved in 20 ml absolutemethanol is irradiated for 2 hours at -30° C. with a 1000 wattsodium-vapour lamp in the presence of 0.01 mol % immobilized Rose Bengalwhile passing in dry oxygen. The reaction proceeds quantitatively. Thesensitizer can be removed by filtration. The filtrate contains thedioxetane.

Yield 2 mmol

EXAMPLE 21 5-Hydroxy-2,2-dimethyl-benzofuran-3-one

80 mmol t-butylhydroquinone together with 80 mmol potassium bromate issuspended in 100 ml water. 20 drops 1 M sulfuric acid is added theretowhile stirring. The reaction mixture is slowly heated to 80° C. whilestirring. A clear orange solution of the quinone forms. When this iscooled to room temperature orange needles precipitate(t-butylbenzoquinone). These are dissolved in 400 ml ethanol while stillwet. The solution is irradiated for 15 h at 5° C. with a 250 Wsodium-vapour lamp (TLC control). The solvent is removed bydistillation, the residue is washed with toluene/hexane 1:1. Theremaining colourless crystalline residue(2-(2'-ethyl-2'-ethoxy-propyl)-hydroquinone) is fed into a mixture of 4ml ethanol and 80 ml glacial acetic acid. 4 ml 40% aqueoustetrafluoroboric acid is added while stirring. The mixture is heated to80° C. during which a clear solution forms. After cooling to roomtemperature it is poured onto 200 ml water and extracted with toluene.The organic phase is washed once with water and dried over potassiumcarbonate. After removing the solvent by distillation an oil remains(5-hydroxy-2,2-dimethyl-2,3-dihydro-benzofurane). The benzoylation,oxidation and saponification are carried out as described in example 15.

Yield 25 mmol

EXAMPLE 22 6-Chloro-5-hydroxy-2,2-dimethyl-benzofuran-3-one

25 mmol 7-Hydroxy-2,2-dimethyl-benzofuran-3-one and 30 g silica gel arestirred in 200 ml chloroform. A solution of 25 mmolN-chloro-diisopropylamine in 100 ml chloroform (prepared fromdiisopropylamine and aqueous sodium hypochlorite solution, extracted inchloroform, concentration determined iodometrically) is added dropwise.After stirring for 10 hours it is filtered and the filtrate is shakenout with water. The solution is dried over sodium sulfate. The solventis removed by distillation at 300 mbar. The residue is separated bycolumn chromatography (silica gel: ethyl acetate).

Yield 20 mmol

EXAMPLE 23 6-Chloro-5-hydroxy-2,2-dimethyl-benzofurane-3-thione

A solution of 20 mmol 6-chloro-5-hydroxy-2,2-dimethyl-benzofuran-3-onein 30 ml toluene is admixed with 10 mmol Lawesson's reagent and heatedfor 3 hours to boiling under reflux. After cooling to room temperatureit is filtered on 50 g silica gel. The red fraction of the filtrate iscollected. After removing the toluene by distillation a red oil remains.

Yield 17 mmol

EXAMPLE 243-((E/Z)-)-6-chloro-5-hydroxy-2,2-dimethyl-benzofuran-3-ylidene)-androsta-1,4-diene-17-one

1 mmol Androsta-1,4-diene-3,17-dione-3-hydrazone is fed while stirringat room temperature into a suspension of 1.5 g manganese dioxide and 1.5g aluminium oxide in 20 ml ether which had been admixed with 10 drops ofwater. After stirring for 1 hour the etheric solution of thediazosteroid is removed by filtration. 1 mmol6-chloro-5-hydroxy-2,2-dimethyl-benzofurane-3-thione is added inportions to the filtrate. After stirring for two hours the ether isremoved by distillation and the residue is chromatographed on silica gel(ethyl acetate/petroleum ether 1:9). The weakly yellow colouredfractions are collected. After removing the solvent by distillation alight yellow crystalline residue remains.

Yield 0.7 mmol

EXAMPLE 253-((E/Z)-)-6-chloro-5-t-butyldimethylsilyloxy-2,2-dimethyl-benzofuran-3-ylidene)-androsta-1,4-diene-17-one

30 mmol3-(androsta-1',4'-diene-17'-one-3'-ylidene)-6-chloro-5-hydroxy-2,2-dimethyl-benzofuraneis suspended in 30 ml absolute methylene chloride. 60 mmol Imidazole and60 mmol t.-butyldimethylsilyl chloride are added in succession. Afterstirring for 14 hours at room temperature it is filtered. The filtrateis successively shaken out with 100 ml 1 M sodium hydroxide solution,100 ml 1 M hydrochloric acid and 20 ml water each time. The separatedorganic phase is dried over sodium sulfate. The solvent is removed bydistillation. The oily residue is separated by column chromatography(silica gel: petroleum ether).

Yield 24 mmol

EXAMPLE 263-((E/Z)-)-6'-Chloro-2',2'-dimethyl-benzofuran-3'-ylidene)-androsta-1,4-diene-17-one-5'-yl-disodiumphosphate

A solution of 30 mmol3-(androsta-1',4'-diene-17'-one-3'-ylidene)-6-chloro-5-hydroxy-2,2-dimethyl-benzofuranein a mixture of 30 ml acetone and 30 ml pyridine is added dropwise whilestirring at 0° C. to a solution of 15 ml phosphoroxy chloride in 75 mlacetone. It is stirred for 1 hour at 0° C. and subsequently it is pouredonto 600 ml saturated sodium chloride and suction filtered. The residueis suspended in 15 ml water. A pH value of 10 is set using 2 M sodiumhydroxide solution. It is admixed with 300 ml ethanol and suctionfiltered. The residue is dried in a vacuum.

Yield 23 mmol

EXAMPLE 27(4'Ξ)-5"-t.-Butyldimethylsilyloxy-6'-chloro-2",2"dimethyl-17-oxo-(3ξ)-dispiro[androsta-1,4-diene-3,3'-[1,2]dioxetane-4',3"-benzofurane

10 mmol3-(androsta-1',4'-diene-17'-one-3'-ylidene)-6-chloro-5-t-butyldimethylsilyloxa-2,2-dimethyl-benzofuranedissolved in 20 ml absolute methylene chloride is irradiated for 2 hoursat -30° C. with a 1000 watt sodium-vapour lamp in the presence of 0.01mol % immobilized Rose Bengal while passing in dry oxygen. The reactionproceeds quantitatively. The sensitizer can be removed by filtration.The filtrate contains the dioxetane.

Yield 20 mmol

EXAMPLE 28(4'Ξ)-6"-chloro-2",2"-dimethyl-17-oxo-(3ξO)-dispiro[androsta-1,4-diene-3,3'-[1,2]dioxetane-4',3"-benzofuran-5"-yl-disodiumphosphate

2 mmol disodium salt of3-(androsta-1',4'-diene-17'-one-3'-ylidene)-6-chloro-5-phosphoryl-2,2-dimethyl-benzofuranedissolved in 20 ml absolute methanol is irradiated for 8 hours at -30°C. with a 1000 watt sodium-vapour lamp in the presence of 0.01 mol %immobilized Rose Bengal while passing in dry oxygen. The reactionproceeds quantitatively. The sensitizer can be removed by filtration.The filtrate contains the dioxetane.

Yield 2 mmol

The corresponding dioxetanes based on the following benzofuranederivatives are prepared analogously to examples 1 to 28:

5-hydroxy-2,2-dimethyl-2,3-dihydro-benzofurane,

5-hydroxy-2,2-dimethyl-2,3-dihydro-benzofuran-3-one,

5-hydroxy-2,2-dimethyl-2,3-dihydro-benzofurane-3-thione,

6-chloro-5-hydroxy-2,2-dimethylbenzofuran-3-one and

6-chloro-5-hydroxy-2,2-dimethyl-benzofurane-3-thione.

What is claimed is:
 1. A compound of formula (la) or (Ib) ##STR8##wherein R¹ and R² are each independently selected from the groupconsisting of hydrogen, straight-chained or branched C₁ -C₆ alkyl,cycloalkyl and aryl, or R¹ and R² together form a cycloalkyl oraryl,with the proviso that at most one of R¹ and R² is hydrogen; R³ is agroup which is capable of being cleaved off the O atom to which it isattached by an activating agent; R⁴ and R⁵ are each independentlyhydrogen or a stabilizing group which protects the dioxetane group fromuncontrolled conversion, or R⁴ and R⁵ together form a stabilizinggroup,with the proviso that at most one of R⁴ and R⁵ is hydrogen; W isselected from the group consisting of hydrogen, halogen andpseudohalogen; X and Y are each independently selected from the groupconsisting of a double bond, a carbonyl group, oxygen, N-R and C(R)₂,wherein R has the meanings of R¹ and R² above, m is 1 or 2; and n is 0or
 1. 2. The compound of claim 1, wherein the compound is selected fromthe group consisting of:a compound of formula (la), wherein X is oxygen,n is 0 and O-R³ is located on position 5 of the phenyl ring; a compoundof formula (Ib), wherein m is 1 and O-R³ is located on position 3 of thephenyl ring; and a compound of formula (Ib), wherein m is 2 and O-R³ islocated on position 4 of the phenyl ring,wherein R¹ -R⁵, W and Y are asdefined above.
 3. The compound of claim 1, wherein O-R³ is selected fromthe group consisting of a hydroxy salt, an oxy acid, a phosphate group,an arylcarboxylic ester, an alkylcarboxylic ester, an alkyloxy group, atrialkyloxy group, an alkylsilyloxy group, an arylsilyloxy group, asulfate group, an oxypyranoside, a glyceridyl group and a phosphorylgroup.
 4. The compound of claim 1, wherein O-R³ is a phosphate group ora dimethyl-tertiary butyl-silyloxy group.
 5. The compound of claim 1,wherein R¹ and R² are each independently selected from the groupconsisting of hydrogen, methyl, ethyl and phenyl which is unsubstitutedor substituted, or R¹ and R², together with the carbon atoms to whichthey are bound, form a phenylene group which is unsubstituted orsubstituted.
 6. The compound of claim 1, wherein R⁴ and R⁵, togetherwith the carbon atoms to which they are bound, form a condensedaliphatic or aromatic ring system which is unsubstituted or has at leastone electron withdrawing group as a substitutent.
 7. The compound ofclaim 6, wherein R⁴ and R⁵ are selected from the group consisting ofadamantanyl group, phenyl group, cyclohexyl group, secondary or tertiaryaliphatic alkyl group, polycycloalkyl group, polycycloaryl group, or asteroid derivative group, wherein any of the groups are unsubstituted orsubstituted with at least one electron withdrawing group.
 8. Thecompound of claim 1, wherein the compound is of formula (la') or (la")##STR9## wherein R¹ -R³ and W are as defined above.
 9. The compound ofclaim 8, wherein the compound is selected from the group consistingof:3-(7'-t.-butyldimethylsilyloxy-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(tricyclor3.3.1.1.³,7]yl)-1,2-dioxetane, a disodium salt of3-(7'-phosphoryl-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(tricyclo[3.3.1.1.³,7 ]yl)-1,2-dioxetane,3-(7'-t.-butyl-dimethylsilyloxy-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7 ]yl)-1,2-dioxetane, a disodium salt of3-(7'-phosphoryl-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7 ]-yl)-1,2-dioxetane,3-(5'-t.-butyldimethylsilyloxy-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7 ]yl)-1,2-dioxetane, a disodium salt of3-(5'-phosphoryl-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7 ]yl)-1,2-dioxetane,3-(5'-t.-butyldimethylsilyloxy-6'-chloro-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7 ]yl)-1,2-dioxetane, a disodium salt of3-(5'-phosphoryl-6'-chloro-2',2'-dimethyl-benzofuran-3'-yl)-4-spiro-(5"-chloro-tricyclo[3.3.1.1.³,7 ]yl)-1,2-dioxetane,(4Ξ-)-5"-t.-butyldimethylsilyloxy-6"-chloro-2",2"-dimethyl-17-oxo-(3.zeta.)-dispiro[androsta-1,4-diene-3,3'-[1,2]dioxetane-4',3"-benzofuraneand(4Ξ)-6"-chloro-2",2"-dimethyl-17-oxo-(3ξ)-dispiro[androsta-1,4-diene-3,3'-[1,2]dioxetane-4',3"-benzofuran-5"-yl-disodiumphosphate.
 10. A method for determining an analyte, comprising reactingthe analyte with a compound as claimed in claim 1 to cleave the group R³therefrom to produce light from the reaction, and thereafter measuringthe produced light to determined the analyte.
 11. The method of claim10, wherein the analyte is selected from the group consisting of anacid, a base, a salt, an enzyme, a DNA, an inorganic or organic catalystand an electron donor.
 12. The method of claim 10, wherein the analyteis an enzyme or a DNA.
 13. The compound of claim 6 wherein at least oneelectron withdrawing group is a group with a positive δ_(p) value.